1. Technical Field
The present invention relates to adeno-associated virus (AAV)-based eucaryotic vectors and uses thereof. Such vectors, for example, may be utilized to down regulate any targeted viral or cellular gene whose sequence is known. Furthermore, the vectors may also be used to cause the expression of proteins.
The present invention specifically includes an adeno-associated (AAV)-based eucaryotic vector which confers intracellular resistance to human immunodeficiency virus, type 1 (HIV-1), and an adeno-associated (AAV)-based eucaryotic vector which confers intracellular resistance to herpes simplex, type 1 (HSV-1) infection.
2. Background Information
The acquired immunodeficiency syndrome (AIDS) is a chronic debilitating illness characterized by immunodeficiency and opportunistic infections of afflicted individuals. Human AIDS is caused by two members of the lentivirus subfamily of retroviruses, the human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2). The natural course of HIV infection in humans is marked by a relentless progression towards end-stage disease over a period of years. Ultimately, high levels of HIV replication and overwhelming opportunistic infections lead to a fatal outcome in most patients. Current estimates indicate that over one million people in the United States may be infected with HIV-1. Thus, recent progress in antiretroviral chemotherapy notwithstanding, novel approaches to the treatment and prevention of AIDS are urgently needed.
Replication of the pathogenic lentivirus, human immunodeficiency virus (HIV-1) involves a highly complex and tightly regulated coordinate expression of virally encoded genes (Varmus (1988), Cullen et al., Cell 58:423-26 (1989) & Rabson). Following entry of the virus into the cell via cell surface receptors which include the helper T lymphocyte determinant CD4 and other as yet undetermined elements (Arthos et al. (1984), Delgleish et al. (1984) & Camerini et al. (1990)) the viral genome is uncoated and reverse transcribed into proviral DNA. Following translocation of the proviral DNA into the nucleus, transcription is initiated under the right cellular environment. Activation of transcription of the HIV genome is complex and highly regulated. Both cellular factors as well as virally encoded proteins regulate gene expression from the HIV long terminal repeat (LTR). The HIV LTR contains the target sequences for both constitutive and inducible host transcriptional activating factors such as SP1, NF1, TF IID and NK-KB (Cullen et al., supra.), the major transactivator of the HIV LTR is virally-encoded tat protein (Rice et al. (1989), Berkhout et al. (1989), Sodroski et al. (1985) & Arya et al. (1985)). Following initial activation of viral transcription, perhaps by cellular factors, the appearance of the multiple spliced early 2 kb species of viral mRNA which encode regulatory proteins tat, rev and nef is observed. Tat is a potent HIV LTR-specific transactivator which markedly increases transcription of all species of HIV messages including itself. The cis-acting target sequence of tat function, TAR, is present in the 5'untranslated leader of all HIV-1 messages and extends from bases 1 to 59 of HIV transcripts, with the sequences between bases 19 to 44 comprising the core TAR sequence. This region is involved in the formation of a stable stem loop structure which is recognized by tat in a sequence-specific and an orientation dependent-manner (Rice et al. (1989) & Berkhout et al. (1989)). This interaction is essential for expression of all HIV-encoded genes and subsequent viral replication. The action of tat results in the accumulation of viral transcripts by a complex mechanism of action, possibly by promoting RNA elongation and/or increasing the rate of initiation. The transition to the intermediate and late phases of HIV replication is mediated by rev, another regulatory protein encoded by the 2 kb early messages. Expression of rev results in the accumulation of singly spliced 4 kb messages encoding env and vif and full length message encoding gag and pol. Rev appears to increase transport of messages out of the nucleus into the cytoplasm for translation, feedback inhibiting itself. Like tat, rev recognizes a complex stable RNA secondary structure, called the rev responsive element (RRE), composed of several stems and loops and occurring within env coding sequences and present in the singly spliced and unspliced messages. The function of the regulatory protein nef is still controversial (Baltimore, Nature 335:395-96 (1988), Venk, & Cullen et al. Cell 58:423-26 (1989)). It has been suggested that it functions to downregulate HIV gene expression, thus promoting latency. Thus, there are several important regulatory elements in the HIV replication cycle which may function as efficient targets for antiviral therapy.
One novel strategy for use in the treatment and prevention of AIDS involves the concept of "intracellular immunization" whereby individual cells (perhaps stem cells) are rendered resistant to virus replication by the stable introduction of DNA sequences that are transcribed into antisense RNA or mRNA that encodes a protein with a dominant negative phenotype (Herskowitz, Nature 329:212-22 (1987) & Baltimore, Nature 335:395-96 (1988)). The effectiveness of dominant negative molecules in blocking target gene functions of impeding virus replication has been demonstrated in a number of different systems (Friedman et al., Nature 335:452-54 (1988), Malim et al., Cell 58:205-14 (1989) & Trono et al., Cell 59:113-20 (1989)). For virus replication in cell culture, much of the work to date has either involved transient expression systems or the exogenous administration of antisense synthetic oligonucleotides that do not confer permanent resistance to viral replication. To impart perpetual "intracellular immunity" to cells that are the targets for virus replication, it may be desirable to contrive a means for the intracellular synthesis of dominant negative molecules that are capable of interfering with critical steps in the virus life cycle. The feasibility of such a strategy depends on many factors including: (i) the ability to deliver and establish stable expression of DNA sequences within the desired cell population; (ii) the efficiency of expression within the target cell without cytotoxic effects; and, (iii) the true effectiveness of dominant negative molecules in reducing or abolishing the burden of infectious virus.
The capability to control (or prevent) HIV infection by the constitutive intracellular expression of molecules designed to inhibit HIV replication would represent a significant advance in the field of AIDS therapy. Thus, the present inventors have initiated efforts to develop a safe, feasible, and effective system for inserting DNA sequences into the host cell genome that might ultimately confer resistance to HIV replication.
Another virus, Herpes simplex virus (HSV), continues to be a major human pathogen which is associated with serious mucocutaneous and visceral infections, particularly in the neonate and immunocomprised host. Although acyclovir therapy has made a major impact upon the overall treatment of HSV infections, it is not curative, and acyclovir-resistant HSV strains are being reported with increasing frequency Marks et al., Rev. Infect. Dis. 11:474-76 (1989) & Sacks et al., Ann. Intern. Med. 111:893-99 (1989)). Thus, other methods are continually being sought to control HSV-1 replication and infection.
Antisense oligonucleotides targeted against areas of critical viral RNA transcripts including the 5'-untranslated region, splice sites, and the polyadenylation signal have demonstrated significant antiviral activities. Treatment of cells with methylphosphonate oligonucleotides targeted to the splice acceptor regions of immediate early (IE) genes ICP 22 and 47 of HSV-1, for example, has resulted in up to 5% inhibition of virus replication and DNA synthesis with minimal effects upon cellular metabolism (Smith et al., Proc. Natl. Acad. Sci. USA, 83:2878-91 (1986) & Kulka et al., Proc. Natl, Acad. USA 86:6868-72 (1989)). However, exogenously administered oligonucleotides require continuous administration to prevent HSV-1 reactivation, and are subject to degradation by cellular nucleases and the vicissitudes of cellular transport.
Herpes simplex virus replication entails a well orchestrated, regulated cascade in which a virion encapsidated, virus-encoded protein (VP16), in association with cellular DNA binding proteins, transactivates virus IE gene promotors. ICP4, an IE gene, is necessary for subsequent transactivation and regulated expression of early and late viral genes. Thus, the VP16 and ICP4 gene products play pivotal roles in HSV lytic infection, and mutations which inactivate them are generally lethal. Hence, they represent ideal targets for potential control of HSV-1 replication. A stable cell line constitutively expressing a carboxy-terminal truncated, noneffector (transdominant) form of VP16 has recently been shown to specifically inhibit HSV-1 replication by 20-40 fold in comparison to control cells. Baltimore has termed this modulation of cellular resistance to vital infection "intracellular immunization," and a number of groups have recently utilized similar methods of develop transdominant inhibitors of human immunodeficiency virus (HIV-1) regulatory proteins.
Adeno-associated virus (AAV), a parvovirus dependent upon adenovirus or herpes virus for full "lytic" infection (Buller et al., J. Virol. 40:241-47 (1981)), offers several advantages as a eucaryotic viral vector among which are lack of cytopathogenicity, wide host range, stability, clonability into bacterial plasmids allowing for easy manipulation, high frequency transduction, and high frequency integration into host cell DNA in the absence of helper virus coinfection (Lebkowski et al., Mol. Cell. Biol. 8:3988-96 (1988) & McLaughlin et al., J. Virol. 62:1963-2 (1988)). AAV may integrate site specifically into host cellular DNA (Kotin et al., Proc. Nat'l. Acad. Sci., USA 87:2211-15 (1990)). This feature would make it unique among currently used eucaryotic viral vectors, and would make it particularly useful as an antisense vector, as vector integration site influences the overall efficacy of antisense modulation of gene expression.
Site specific integration also greatly reduces the chance of insertional mutagenesis, which is known to occur with retroviral vectors. Other AAV vectors have been described, but none incorporate the features of the present vectors or were designed for the purposes described herein.
All patents and publications referred to herein are hereby incorporated by reference.